1. Field of the Invention
The present invention relates to an immunodiagnostic assay method and kit to rapidly evaluate immunization status of a patient, or alternatively, to verify exposure to a biowarfare agent, such as anthrax, in a patient.
2. Description of the Related Art
A critical need exists for rapid, reliable, non-invasive and safe testing procedures to evaluate immunization status, such as anthrax immunization status, of a patient.
The terrorist attacks of Sep. 11, 2001 in the United States and the outbreak of anthrax in Florida, New York, Washington D.C., etc. shortly thereafter has brought the threat of biowarfare into sharp focus for the general public. Biowarfare agents have been of significant concern for the Department of Defense (DoD) for the last couple of decades in particular. Anthrax is considered one of the most dangerous of biowarfare threats because of its potency and very high mortality rate. One gram of anthrax material is estimated to give 100 million lethal doses, or is 100,000 times more lethal than the deadliest chemical warfare agent. DoD (1998) Information Paper: The Anthrax Vaccine. In Vol. 2001. Anthrax weapons are also relatively inexpensive to develop because the bacterial spores are relatively easy to obtain and can be packaged into a bomb or missile for delivery. It has been estimated that at least 10 countries in the world have, or are developing, biological warfare capability that includes anthrax. Miller, K. (2001) Shots in the Dark. In Army Times pp. 14-16.
The Persian Gulf War (1990-1991) brought the threat of this disease into sharp focus for the DoD, because Iraq was thought to have developed and stockpiled anthrax weapons. As a preventive measure, nearly all British troops and about 150,000 U.S. troops were administered anthrax vaccines for protection in the early stages of the war. To counter the growing threat to military personnel deployed overseas, the Secretary of Defense mandated in 1997 that all active duty personnel must be vaccinated for protection against possible anthrax exposure.
Although the mandatory Anthrax Vaccine Immunization Program (AVIP) was initiated in early 1998, there have been no known follow-up studies in immunized personnel to verify antibody production against the antigenic component of the anthrax toxin. As a result, DoD has sponsored research for developing countermeasures to biological warfare agents and for developing methods of verifying vaccine effectiveness. As part of the DoD Broad Agency Announcement (BAA) 99-1 released in December 1998, the Medical Biological Defense Research Program outlined the need for identification of markers of protection and assay development to assess the level of protection against biological warfare agents.
Current testing methods measure serum antibody levels in response to vaccines by time-consuming, labor-intensive processes. Highly trained laboratory personnel are needed to perform these current tests, and the instrumentation required for current tests is relatively sophisticated and expensive. While Enzyme-Linked Immunosorbent Assay (ELISA) has been the standard for measuring antibody titers, it is more problematic to perform this diagnostic test in forward-deployed regions where military personnel are at greater potential risk for anthrax toxin exposure.
The following discussion provides background on the anthrax bacteria, anthrax vaccines, animal models and anthrax strains.
Anthrax Bacteria Characterization
The Gram-positive, rod-shaped bacterium Bacillus anthracis is the toxic agent of anthrax infection, a natural disease endemic to grazing animals including cattle, goats and sheep. There are three forms of anthrax infection in patients: (1) cutaneous infection that occurs by dermal absorption of the bacteria through a cut or abrasion in the skin; (2) gastrointestinal infection resulting from ingestion of contaminated meat products; and (3) inhalation of anthrax spores, which is the most lethal form of exposure. The prescribed medical treatment for all forms of the disease is high doses of antibiotic, most commonly penicillin. However, bacterial resistance to third-generation cephalosporins has been documented, further thwarting attempts to quickly treat this infection. Shafazand, S., Doyle, R., Ruoss, S., Weinacker, A., and Raffin, T. A. (1999) Inhalational anthrax: epidemiology, diagnosis, and management. Chest 116, 1369-1376.
Fortunately, anthrax is not highly contagious among animals or patients, with infection occurring only by direct absorption of the bacteria in one of the ways listed. However, outbreaks of the disease still occur in countries that do not have active livestock vaccination programs or effective patient vaccines. Therefore, anthrax is still a danger to public health in less-developed countries such as India, where a deadly outbreak occurred in 1999-2000 in Pondicherry territory. See medscape.com (2001) Health experts disturbed at resurgence of anthrax in India. In Vol. 2001, Medscape.
Bacillus anthracis bacteria possess two virulence factors, the anthrax toxin and the protective capsule. The pXO1 plasmid is known to contain the specific genes for the toxin protein components, and the pXO2 plasmid encodes formation of the poly-D-glutamic acid capsule that prevents cellular phagocytic action against the bacteria. Toxins secreted by this bacterium are composed of binary combinations of three proteins: protective antigen (PA), which is only virulent when bound with either one of two additional proteins, lethal factor or edema factor. The primary antigenic protein recovered from bacteria culture filtrates is PA, an 83 kilodalton (kDa) protein encoded by the pag gene on the pXO1 plasmid of the bacterium. Edema factor (EF, 89 kDa), encoded by a separate gene on the pXO1 plasmid, is a calcium/calmodulin-dependent adenylate cyclase that stimulates cyclic adenosine monophosphate (cAMP) production resulting in inflammation, redness and soreness. Lethal factor (LF, 83 kDa), also encoded by a gene on this same plasmid, appears to be a metalloendopeptidase that cleaves the N-terminus of mitogen-activated protein kinase 1 and 2 (MAPKK1 and MAPKK2) and thereby inhibits the MAPK signal transduction pathway.
It is the PA-LF bound form of the toxin that results in serious illness or death if not promptly treated. Brossier, F., Weber-Levy, M., Mock, M., and J-C., S. (2000) Role of toxin functional domains in anthrax pathogenesis. Infect. Immun. 68, 1781-1786.
Actual toxic activity occurs when PA, the binding domain of anthrax toxin, recognizes and binds to cell-surface receptors. This is followed by cleavage of a 20 kDa fragment from the N-terminus by furin-like cellular proteases, leaving the membrane-bound, mature PA protein (63 kDa). Proteolytic activation of PA causes formation of a membrane-inserting heptamer that creates a channel through the cell membrane. This activation process also exposes a high affinity binding site on PA for which the other two proteins LF and EF compete. After either factor binds to PA, the protein complex then translocates through the membrane via receptor-mediated endocytosis to deliver the toxic enzymes to the cell. Once in the cytoplasm, these toxins disrupt normal cellular functions, eventually causing cell death.
Several laboratories have shown that specific critical components of the PA protein confer the toxic nature of anthrax. Singh et al. have shown that the C-terminus of PA is crucial to recognition of and binding by the host cell. Singh, Y., Klimpel, K. R., Quinn, C. P., Chaudhary, V. K., and Leppla, S. H. (1991) The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity. J. Biol. Chem. 266, 15493-15497.
By constructing truncations of the C-terminal domain or deletion mutants in the C-terminus loop, different laboratories were able to create a form of anthrax that is less cytotoxic or nontoxic. Singh, Y., Klimpel, K. R., Quinn, C. P., Chaudhary, V. K., and Leppla, S. H. (1991) The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity. J. Biol. Chem. 266, 15493-15497; and Brossier, F., Sirard, J.-C., Guidi-Rontani, C., Duflot, E., and Mock, M. (1999) Functional analysis of the carboxy-terminal domain of Bacillus anthracis protective antigen. Infect. Immun. 67, 964-967.
Another laboratory showed that pretreatment with anti-PA monoclonal antibodies prevented binding of 125I-LF or 125I-EF labeled analogs to activated PA, thereby inhibiting toxin activity. Little, S. F., Novak, J. M., Lowe, J. R., Leppla, S. H., Singh, Y., Klimpel, K. R., Lidgerding, B. C., and Friedlander, A. M. (1996) Characterization of lethal factor binding and cell receptor binding domains of protective antigen of Bacillus anthracis using monoclonal antibodies. Microbiology 142, 707-715; and Little, S. F., Ivins, B. E., Fellows, P. F., and Friedlander, A. M. (1997) Passive protection by polyclonal antibodies against Bacillus anthracis infection in guinea pigs. Infect Immun 65, 5171-5175.
Petosa demonstrated that the first, or N-terminal domain of PA, containing the protease cleavage site, is also susceptible to mutation. When point mutations were made in the amino acid sequence of this region, cleavage of the 20 kDa N-terminus did not occur and PA was not activated to expose the EF/LF binding site, resulting in no toxicity. Petosa, C., Collier, R. J., Klimpel, K. R., Leppla, S. H., and Liddington, R. C. (1997) Crystal structure of the anthrax toxin protective antigen. Nature 385, 833-838.
This spore-forming bacterium is also widely considered a possible weapon for biological warfare and bioterrorism because the spores are extremely stable and robust, surviving for many years in arid and semi-arid conditions. Shafazand, S., Doyle, R., Ruoss, S., Weinacker, A., and Raffin, T. A. (1999) Inhalational anthrax: epidemiology, diagnosis, and management. Chest 116, 1369-1376.
In the spore form, Bacillus anthracis may easily be dispersed over a wide area and poses a lethal challenge when inhaled. The DoD estimates that among exposed individuals, the lethal dose (LD50) is between 8,000 and 10,000 spores. Block, S. M. (2001) The Growing Threat of Biological Weapons. American Scientist 89, 28-37.
Further complications result in that exposure to inhalational anthrax is not easily diagnosed because the infection initially presents with nonspecific influenza-like symptoms. The U.S. government believes that a number of countries have developed anthrax biological weapons and are capable of releasing these weapons. Concern for public safety from possible terrorist acts has led many cities to develop an emergency contingency plan outlining medical and civic responses in the event of a toxin release. The possibility of chemical or biological agent exposure may not be so unimaginable in that a 1995 terrorist release of sarin nerve gas in a crowded Tokyo subway had a devastating outcome, affecting almost 5,000 people.
Anthrax Vaccine History
In the 1870's Louis Pasteur studied the bacterium Bacillus anthracis in some of his earliest work in bacteriology and the germ theory of disease. Brachman, P. S. (1970) Anthrax. Ann. NY Acad. Sci. 174, 577-582. Robert Koch also used this bacterium in 1876 as a model in describing his postulates of germ theory. Id.
In 1881 Pasteur successfully field-tested an animal anthrax vaccine, a remarkable achievement that set the stage for significant advances in immunology and the understanding and treatment of disease. Id.; Block, S. M. (2001) The Growing Threat of Biological Weapons. American Scientist 89, 28-37; Tumbull, P. C. B. (1991) Anthrax vaccines: past, present and future. Vaccine 9, 533-539.
A serious outbreak of patient anthrax occurred in the United States around 1924 and again in the early 1930's. At that time, cutaneous and inhalation anthrax occurred at a high rate among individuals working in textile mills and animal processing plants who handled the hides and hair of infected cattle, sheep and goats both domestic and imported. This significant health risk led to the first animal anthrax vaccination program and to the development of a patient anthrax vaccine in the 1950's. As a result of the vaccination program for workers at risk, the incidence of anthrax endemic to the U.S. virtually disappeared over the next four decades. The vaccine was reformulated during the period 1957-1960 to select a strain of anthrax that produced a higher fraction of PA. A form of protein-free culture medium was also employed and aluminum hydroxide was adapted as the adjuvant. Ashford, D. A., D. V. M., M. P. H., D. Sc., and Rotz, L. D., M.D. (2000) Use of Anthrax Vaccine in the United States. In pp. 1-20, Centers for Disease Control and Prevention, Atlanta, Ga.
This vaccine was licensed for patient use in 1970 by the National Institutes of Health, and was later approved by the U.S. Food and Drug Administration (FDA). DoD (1998) Information Paper: The Anthrax Vaccine. In Vol. 2001.
In 1990, filtration-sterilization procedures in the anthrax vaccine production process were changed, resulting in higher levels of PA toxin component in the vaccine. Ivins, B. E., Fellows, P. F., and Nelson, G. O. (1994) Efficacy of a standard patient anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12, 872-874.
Current Vaccine Development
In the United States, BioPort Corporation in Lansing, Mich. exclusively produces the anthrax vaccine currently used for patients. The Anthrax Vaccine Adsorbed (AVA) vaccine is produced from the antigenic protein PA, recovered from cell-free culture filtrates of an avirulent, non-encapsulated strain of Bacillus anthracis. The filtrate is adsorbed onto aluminum hydroxide, an adjuvant that stimulates antibody production (humoral response) against the antigenic toxin protein. Currently, anthrax immunization involves six injections over an 18 month period, with annual boosters thereafter. The first three subcutaneous injections are given 2 weeks apart, followed by three additional injections at 6, 12 and 18 months. This immunization schedule is based upon one determined for earlier generations of anthrax vaccine and studies that examined vaccine effectiveness in animal models at that time, but additional work is needed to determine efficacy of recently developed vaccines.
Presently, there is little direct scientific evidence that patients vaccinated with AVA are protected against anthrax. The only patient study that addresses vaccine effectiveness was an epidemiological evaluation of vaccinated and unvaccinated employees in four textile mills in the 1950's and the incidence of anthrax among these at-risk workers. Brachman, P. S., Gold, H., Plotkin, S. A., Fekety, F. R., Werrin, M., and Ingraham, N. R. (1962) Field evaluation of a patient anthrax vaccine. Am. J. Public Health 52, 632-645.
The anthrax vaccine in use at the time predated the AVA that was reformulated for patient use around 1960. More documentation exists for vaccine protection against cutaneous anthrax, the most common form of the disease (reported to be greater than 95% of all U.S. cases (Brachman, P. S. (1970) Anthrax. Ann. NY Acad. Sci. 174, 577-582), with very little proof of effectiveness against the inhalation form of infection. Other indirect evidence of vaccine effectiveness was gathered in a retrospective analysis that measured patient IgG antibody titer after AVA administration in stored sera of immunized laboratory personnel. Pittman, P. R., Mangiafico, J. A., Rossi, C. A., Cannon, T. L., Gibbs, P. H., Parker, G. W., and Friedlander, A. M. (2000) Anthrax vaccine: increasing intervals between the first two doses enhances antibody response in patients. Vaccine 19, 213-216.
Animal Models and Anthrax Strains
Several experimental animal models have been studied to assess the effectiveness of patient AVA vaccine in the United States and of the vaccine used in the United Kingdom. However, the two vaccines differ in the Bacillus anthracis strain used. Historically, guinea pigs have been the primary animal model studied to evaluate vaccine effectiveness. Results of studies conducted with this model show that AVA gives variable protection when the animals are challenged intramuscularly with anthrax spores. In these studies, 0% to 100% of the immunized animals survived challenge with various spore strains. Ivins, B. E., Fellows, P. F., and Nelson, G. O. (1994) Efficacy of a standard patient anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12, 872-874; Turnbull, P. C. B., Broster, M. G., Carman, J. A., Manchee, R. J., and Melling, J. (1986) Development of antibodies to protective antigen and lethal factor components of anthrax toxin in patients and guinea pigs and their relevance to protective immunity. Infect. Immun. 52, 356-363; Little, S. F., and Knudson, G. B. (1986) Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun 52, 509-512; Ivins, B. E., Welkos, S. L., Little, S. F., Crumrine, M. H., and Nelson, G. O. (1992) Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants. Infect Immun 60, 662-668; and Fellows, P. F., Linscott, M. K., Ivins, B. E., Pitt, M. L. M., Rossi, C. A., Gibbs, P. H., and Friedlander, A. M. (2001) Efficacy of a patient anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19, 3241-3247.
Also, AVA did not provide good protection in the guinea pig against an aerosol spore challenge, with only 20%-26% of the animals surviving. Ivins, B., Fellows, P., Pitt, L., Estep, J., Farchaus, J., Friedlander, A., and Gibbs, P. (1995) Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs. Vaccine 13, 1779-1784.
These studies, although consistent among themselves, demonstrate the tremendous variability of survival rates in this animal model when challenged with different spore strains. There may also be species-specific differences in that the guinea pig has an acute susceptibility to infection, but relative resistance to toxin effects. McBride, B. W., Mogg, A., Telfer, J. L., Lever, M. S., Miller, J., Turnbull, P. C. B., and Baillie, L. (1998) Protective efficacy of a recombinant protective antigen against Bacillus anthracis challenge and assessment of immunological markers. Vaccine 16, 810-817.
It was also proposed that this animal model may respond poorly to the AVA vaccine itself. Fellows, P. F., Linscott, M. K., Ivins, B. E., Pitt, M. L. M., Rossi, C. A., Gibbs, P. H., and Friedlander, A. M. (2001) Efficacy of a patient anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19, 3241-3247.
This observation is supported by studies in which PA vaccines with different adjuvants caused higher antibody titers against PA than AVA and seemed to confer better protection. Ivins, B. E., Welkos, S. L., Little, S. F., Crumrine, M. H., and Nelson, G. O. (1992) Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants. Infect Immun 60, 662-668.
The original Steme strain (pXO1+, pXO2+) has proven to be the most virulent in guinea pigs, while the Ames and Vollum 1B strains showed progressively decreasing levels of virulence. A few of the studies cited above employed challenges with different strains of anthrax spores, thereby introducing the possibility that the variable survival rates were due to the virulence of the spore strain. In these studies, it was shown that patient vaccine provided significantly better protection in animals challenged with Vollum or Vollum 1B spore strains than those challenged with the Ames strain (also known as a ‘vaccine resistant’ strain). Turnbull, P. C. B., Broster, M. G., Carman, J. A., Manchee, R. J., and Melling, J. (1986) Development of antibodies to protective antigen and lethal factor components of anthrax toxin in patients and guinea pigs and their relevance to protective immunity. Infect. Immun. 52, 356-363; Little, S. F., and Knudson, G. B. (1986) Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun 52, 509-512; and Ivins, B. E., Fellows, P. F., and Nelson, G. O. (1994) Efficacy of a standard patient anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12, 872-874.
This suggests that virulence of the challenge anthrax strain also impacts the degree of protection provided by patient vaccines. Alternately, a study of immunization with a live Sterne spore vaccine resulted in a higher survival rate than in those animals immunized with the PA-based patient vaccine and challenged with different spore strains. Turnbull, P. C. B., Broster, M. G., Carman, J. A., Manchee, R. J., and Melling, J. (1986) Development of antibodies to protective antigen and lethal factor components of anthrax toxin in patients and guinea pigs and their relevance to protective immunity. Infect. Immun. 52, 356-363; Little, S. F., and Knudson, G. B. (1986) Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun 52, 509-512.
In studies examining survival rates of immunized guinea pigs challenged intramuscularly with the Ames spore strain, anti-PA antibody titers were also measured and found to be an accurate predictor of survival in one study (see Barnard, J. P., and Friedlander, A. M. (1999) Vaccination against anthrax with attenuated recombinant strains of Bacillus anthracis that produce protective antigen. Infect. Immun. 67, 562-567), but not an accurate predictor in other studies. See Turnbull, P. C. B., Broster, M. G., Carman, J. A., Manchee, R. J., and Melling, J. (1986) Development of antibodies to protective antigen and lethal factor components of anthrax toxin in patients and guinea pigs and their relevance to protective immunity. Infect. Immun. 52, 356-363; Little, S. F., and Knudson, G. B. (1986) Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun 52, 509-512; Ivins, B. E., Welkos, S. L., Little, S. F., Crumrine, M. H., and Nelson, G. O. (1992) Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants. Infect Immun 60, 662-668; and Ivins, B. E., Fellows, P. F., and Nelson, G. O. (1994) Efficacy of a standard patient anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12, 872-874.
The latter studies found no significant correlation between anti-PA antibody titer and survival using patient vaccines isolated from culture filtrates. However, it should also be noted in studies conducted before 1990, the patient vaccine used was not optimized for PA expression. The type of adjuvant used also influenced the protective effect in experimental vaccines (see Ivins, B. E., Fellows, P. F., and Nelson, G. O. (1994) Efficacy of a standard patient anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12, 872-874 Ivins, B. E., Fellows, P. F., and Nelson, G. O. (1994) Efficacy of a standard patient anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12, 872-874) and affected the amount of anti-PA produced against the vaccine (see McBride, B. W., Mogg, A., Telfer, J. L., Lever, M. S., Miller, J., Turnbull, P. C. B., and Baillie, L. (1998) Protective efficacy of a recombinant protective antigen against Bacillus anthracis challenge and assessment of immunological markers. Vaccine 16, 810-817).
A recent study using rabbits supports the conclusion that antibody levels to PA after AVA immunization predicted protection in animals receiving an aerosolized Ames spore challenge. Pitt, M. L. M., Little, S., Ivins, B. E., Fellows, P., Boles, J., Barth, J., Hewetson, J., and Friedlander, A. M. (1999) In vitro correlate of immunity in an animal model of inhalational anthrax. J Appl. Microbiol. 87, 304.
A protective effect of 90% or greater was also realized in AVA-immunized rabbits challenged with a number of virulent spore strains, confirming efficacy of the vaccine in this animal model. Fellows, P. F., Linscott, M. K., Ivins, B. E., Pitt, M. L. M., Rossi, C. A., Gibbs, P. H., and Friedlander, A. M. (2001) Efficacy of a patient anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19, 3241-3247.
It is the work conducted with non-patient primates, however, that is believed to most closely predict the protection afforded patients against anthrax spore inhalation. In a study conducted with rhesus macaques immunized with patient AVA (and with other experimental vaccines using rPA, a recombinant form of PA), all vaccinated monkeys survived a subsequent aerosol challenge of lethal doses of Ames strain spores, while all control animals perished. Ivins, B. E., Pitt, M. L., Fellows, P. F., Farchaus, J. W., Benner, G. E., Waag, D. M., Little, S. F., Anderson, G. W. J., Gibbs, P. H., and Friedlander, A. M. (1998) Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques. Vaccine 16, 1141-1148.
In a recent study in which AVA protection of guinea pigs, rabbits and rhesus macaques was evaluated, rabbits and non-patient primates showed nearly complete protection against an aerosol spore challenge. Fellows, P. F., Linscott, M. K., Ivins, B. E., Pitt, M. L. M., Rossi, C. A., Gibbs, P. H., and Friedlander, A. M. (2001) Efficacy of a patient anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19, 3241-3247.
Other laboratories have shown that vaccines containing PA derived from various recombinant plasmids are effective against aerosolized toxin exposure in nonpatient primates (see Ivins, B. E., Pitt, M. L., Fellows, P. F., Farchaus, J. W., Benner, G. E., Waag, D. M., Little, S. F., Anderson, G. W. J., Gibbs, P. H., and Friedlander, A. M. (1998) Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques. Vaccine 16, 1141-1148) or in other animal models. See Zaucha, G. M., Pitt, M. L. M., Estep, J., Ivins, B. E., and Friedlander, A. M. (1998) The pathology of experimental anthrax in rabbits exposed by inhalation and subcutaneous inoculation. Arch. Pathol. Lab. Med. 122, 982-992; Barnard, J. P., and Friedlander, A. M. (1999) Vaccination against anthrax with attenuated recombinant strains of Bacillus anthracis that produce protective antigen. Infect. Immun. 67, 562-567; Pitt, M. L. M., Little, S., Ivins, B. E., Fellows, P., Boles, J., Barth, J., Hewetson, J., and Friedlander, A. M. (1999) In vitro correlate of immunity in an animal model of inhalational anthrax. J. Appl. Microbiol. 87, 304; and Shafazand, S., Doyle, R., Ruoss, S., Weinacker, A., and Raffin, T. A. (1999) Inhalational anthrax: epidemiology, diagnosis, and management. Chest 116, 1369-1376.
Based upon this large body of evidence, a new experimental vaccine using rPA as the antigenic component is now under development at a number of laboratories. When approved by the FDA, this new generation of anthrax vaccines will become the standard to immunize personnel at risk of exposure.
The variability of survival outcomes in animal models may be a direct effect of the differing virulence of the challenge strains and the types of vaccines used. Therefore, the results are inconclusive because vaccine efficacy in one animal model cannot be compared to the protection afforded other animals immunized with the same vaccines or challenged with the same anthrax strains. This also clearly points out the inherent difficulty in extrapolating results of anthrax vaccine protection in animals to that in patients. In the absence of any controlled patient trials of vaccine effectiveness, the best animal model is the non-patient primate. Clearly, additional work needs to be conducted with this model.